Microbial swollenin protein, dna sequences encoding such swollenins and method of producing such swollenins

ABSTRACT

A novel microbial protein is described which appears to have significant homology to plant expansin proteins and has the ability to weaken filter paper and swell cellulose. A DNA is described which encodes the novel protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/893,766, filed Jul. 11, 1997 and which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Osmotic uptake of water is the driving force of plant cellexpansion. As water enters the cell, the protoplast expands but isrestrained by the cell wall. Moreover, a rigid complex of cellulosemicrofibril polymers embedded in a glue-like matrix of pectins,hemicelluloses and proteins forms part of this wall in mature cells. Ithas long been thought that some “wall loosening” factor must be presentwhich alters immature cell wall mechanical properties and allows it toundergo a process of elongation. McQueen-Mason et al., Plant Cell, Vol.4, pp. 1425-1433 (1992) studied plant cell enlargement regulation byemploying a reconstitution approach. The authors found that a crudeprotein extract from the cell walls of growing cucumber seedlingspossess the ability to induce the extension of isolated cell walls.Sequential HPLC fractionation of the active wall extract revealed twoproteins with molecular masses of 29 and 30 kD associated with theactivity. Each protein, by itself, could induce wall extension withoutdetectable hydrolytic breakdown of the wall and appeared to mediate“acid growth” responses of isolated walls and may catalyze plant cellwall extension by a novel biochemical mechanism.

[0003] Shcherban et al., Proc. Nat. Acad. Sci., USA, Vol. 92, pp.9245-9249 (1995) isolated cDNA's encoding these two cucumber proteinsand compared them to anonymous expressed sequence tags from varioussources. Rice and Arabidopsis expansin cDNA were identified from thesecollections and showed at least four different expansin cDNA's in riceand six different expansin cDNA's in Arabidopsis. The authors concludedteat expansin are highly conserved in size and sequence (60-87% aminoacid identity and 75-95% similarity between any pairwise comparison) andthat the multigene family formed before the evolutionary divergencebetween monocotyledons and dicotyledons. Shcherban et al. states thatthe high conservation of this multigene family indicates that themechanism by which expansin promotes cell wall extension tolerateslittle variation in protein structure.

[0004] Wang et al., Bioteh. Lett., Vol. 16, No. 9, pp. 955-958 (1994)discovered two proteins in a Chinese medicinal cucumber, Trichosantheskirilowii, which appear to be similar to the S1 and S2 proteins whichdemonstrate cell wall extension properties. Similar proteins were alsofound in growing tomato leaves (Keller et al., The Plant Journal, Vol.8, No. 6, pp. 795-802 (1995)) and in oat coleoptile walls (Li et al.,Planta, Vol. 191. pp. 349-356 (1993)).

[0005] Cosgrove et al., J. Exp. Botany, Vol. 45, Special Issue, pp.1711-1719 (1994) suggested that cooperative interactions between theexpansin proteins and pectinases and cellulases may occur, wherein theenzymes modify the matrix so that other wall extension mechanisms may bemore effective. Fry, Current Biology, Vol. 4, No. 9 (1994) suggest that,in loosening cell walls, expansin seems unlikely to breakcellulose-cellulose bonds as microfibrils remain intact during growth.Thus, the authors discount the observed breakage of hydrogen bonds infilter paper as a side issue and suggest that expansin may lengtheninter-microfibrillar tethers by causing hemicellulose chains to detachfrom cellulose microfibrils to allow extension.

[0006] Despite the pioneering work previously done in the area of cellwall extension and its causes, work related to the usefulness andoperability of expansins is still in its infancy. Moreover, the sourcesof expansin up to now have been exclusively from plant origins, forwhich expression systems may not be optimal for large scale production.Accordingly, it would be valuable to have a ready source ofexpansin-like material which is capable of being produce in largequantities from organisms which are established high output producers ofbiological materials, such as fungi, bacteria or other wellcharacterized microorganisms.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide for aswollenin protein which is derived from a microbial non-plant source.

[0008] It is another object of the present invention to provide for aswollenin protein which is expressible in a well-characterizedmicroorganism, for example a fungus or bacteria, so as to facilitate itsproduction in large quantities.

[0009] It is yet another object of the present invention to provide aDNA sequence corresponding to a microbial swollenin which can be used inindustrial production of swollenin protein.

[0010] It is yet another object of the present invention to provide fornovel and useful methods of altering cellulosic substrates, such as pulpand paper, cellulose based textile fibers, animal feed and corn wetmilling or dry milling polysaccharide waste products or other cellulosicbiomass.

[0011] According to the present invention, a partially or whollyisolated swollenin protein derived from a fungus or bacteria isprovided. Preferably, the swollenin is derived from a filamentousfungus, more preferably, from a filamentous fungus such as Trichodermaspp., Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp.,Penicillium spp., or Gliocladium spp. and most preferably, fromTrichoderma spp. In a particularly preferred embodiment of the presentinvention, the swollenin comprises a sequence according to SEQ. ID NO:2,has at least 70% sequence identity with the sequence provided in SEQ. IDNO:2 or comprises a derivative of the sequence according to SEQ. IDNO:2, wherein the swollenin further has the ability to weaken filterpaper and/or swell cotton fibers.

[0012] In another embodiment of the present invention, a DNA is providedencoding a swollenin protein from a fungus or bacteria. Preferably, theDNA is derived from a filamentous fungus such as Trichoderma spp.,Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp.,Penicillium spp., or Gliocladium spp. Also preferably, the DNA comprisesthe sequence according to SEQ. ID. NO:1. Alternately, the DNA has atleast 70% sequence identity with the sequence according to SEQ. ID NO: 1or comprises a derivative of the sequence according to SEQ. ID NO:1,wherein said DNA encodes a swollenin protein which has the ability toweaken filter paper and/or swell cotton fibers. In a preferredembodiment of the invention, the DNA hybridizes with a DNA having all orpart of the sequence provided in SEQ ID NO:1.

[0013] In another embodiment of the invention, a DNA is provided whichencodes a microbial, e.g., bacterial or fungal, swollenin, and the DNAhybridizes with a DNA probe encoding a peptide having an amino acidsequence comprising SEQ. ID NO:14, SEQ. ID NO:15, SEQ. ID NO:16, SEQ. IDNO:17 or SEQ. ID NO:18. Vectors comprising such DNA, host cells havingbeen transformed with such vectors and fermentation broths produced bysuch transformed host cells are also within the scope of the presentinvention.

[0014] In yet another embodiment of the present invention, a method ofproducing swollenin protein is provided comprising the steps of (a)obtaining a host cell which has been transformed with a vectorcomprising DNA encoding a swollenin protein, the DNA being isolated froma fungus or bacteria; (b) culturing the host cell under conditionssuitable for the expression and, optionally, secretion, of the swolleninprotein; and (c) recovering the fermentation broth containing saidswollenin protein.

[0015] Since fungi and bacteria do not generally have a cellulosic cellwall and in any event are not known to increase in size by the samemechanism as higher plants, Applicants discovery that thesemicroorganisms produce proteins having expansin-like properties is notsuggested by previous work related to plant expansins. Thus, the findingthat the cellulclytic fungus Trichoderma spp. produces an expansin-likeprotein is unexpected. However, it is apparent that the microbial classof proteins differs from those heretofore discovered in plants. Forexample, the presence of a region on the microbial swollenin proteindescribed herein corresponding to the cellulose binding domain of fungalcellulclytic enzymes suggests that this protein is secreted to act inconcert with the naturally secreted cellulases and hemicellulases inorder to facilitate hydrolysis of cellulosic biomass in the environment.Consistent with this suggestion, the Trichoderma reesei swollenin genewas found to be expressed when the fungus was grown on cellulose as asole carbon source, but not when the carbon source for growth wasglucose. This pattern of regulation of gene expression is similar tothat observed for many of the Trichoderma cellulose and hemicellulosegenes. These unexpected findings lead to the conclusion that celluloseor hemicellulose degrading micro-organisms, including bacteria, yeastand fungi, would also produce such swollenin proteins.

[0016] Accordingly, it is an advantage of the present invention that theswollenins provided herein may have utility in many applications forwhich cellulase is currently used, for example, cleaning textiles(laundry detergents and pre-wash compositions), modifying textiles(depilling, color restoration, anti-greying), stonewashing denim,biomass conversion to glucose, and improvement of the nutritive value ofanimal feeds. Similarly, it is contemplated that an advantage of thepresent invention is that swollenins may have a synergistic or additiveeffect in combination with other enzymes, particularly cellulases suchas endoglucanases. In other cases, it is possible that swollenins wouldhave a deleterious effect in an application; for example, they may causeexcessive fabric strength loss when present as a side activity in anendoglucanase produced by fermentation of a microorganism and used forfabric cleaning or modification. In such a case, removal of theswollenin from a cellulase product may be beneficial and may beaccomplished by biochemically removing the product from the resultantcellulase mixture, through genetic engineering to prevent its expressionor to inactivate the gene or by adding a chemical inhibitor to thecomposition comprising the swollenin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 illustrates the nucleotide sequence (SEQ ID NO:1) andpredicted corresponding amino acid sequence (SEQ ID NO:2) of a cDNAclone obtained from a Trichoderma reesei (longibrachiatum) RNA aftergrowth on a mixed carbon source.

[0018]FIG. 2 illustrates a comparison of the consensus amino acidsequence for plant expansin proteins (SEQ ID NO:3) and the sequence ofthe swollenin (SEQ ID NO:4) described herein showing the regions ofamino acid homology.

[0019]FIG. 3 illustrates the result of Northern blotting of RNA samplesprepared from Trichoderma reesei (longibrachiatum) mycelium grown ondifferent carbon sources and probed with swollenin cDNA. Lane 1:cellulose; lane 2: glucose; lane 3: sorbitol; lane 4: sorbitol cultureinduced by sophorose.

[0020]FIG. 4 illustrates a comparison of nine known plant expansin aminoacid sequences (SEQ ID NOS:5-13) showing the extensive homology presentin plant expansins.

[0021]FIG. 5 shows the plasmid map for pGAPT-exp.

[0022]FIG. 6 illustrates the results of an SDS-PAGE gel run with culturesupernatants and controls. Aspergillus transformants which wereproducing the T. reesei swollenin have a band running above the 66 kDmarker band and this band is missing from lanes of the negative control(Aspergillus strain before the transformation).

DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] “Swollenin” mean a protein or polypeptide or domain of a proteinor polypeptide of microbial, i.e., fungal or bacterial, origin which hasthe ability to facilitate weakening of filter paper and the swelling ofcotton fibers without having cellulclytic activity, i.e., catalyticactivity involving the breakage of individual cellulose strands intosmaller monomer (glucose) or oligomers (polysaccharides). While it isuseful to define swollenins loosely in terms of the expansin proteinsdescribed in McQueen-Mason et al., Plant Cell, Vol. 4, pp. 1425-1433(1992), it is also apparent that microbial swollenins have distinctproperties, for example, microbial swollenins are much larger proteinsthan plant expansins and have a low level of sequence identity withplant expansins. Moreover, certain microbial swollenin proteins exist inconjunction with a cellulose binding domain and may further exist inconjunction with a catalytic cellulase domain. For example, theswollenin protein derived from Trichoderma reesei shown herein possessesa cellulose binding domain.

[0025] It is contemplated herein that swollenins may be derived frommicrobial origins, and particularly from fungal or bacterial origins.Specifically, it is contemplated that microorganisms which possesscellulolytic capabilities will be excellent sources of swolleninprotein. In a particularly preferred embodiment of the invention, theswollenin is derived from Trichoderma spp., particularly Trichodermareesei (longibrachiatum). However, also preferably, the swollenin and/orDNA encoding swollenin according to the present invention is derivedfrom a fungus, such as, Absidia spp.; Acremonium spp.; Agaricus spp.;Anaeromyces spp.; Aspergillus spp., including A. auculeatus, A. awamori,A. flavus, A. foetidus, A. fumaricus, A. fumigatus, A. nidulans, A.niger, A. oryzae, A. terreus and A. versicolor; Aeurobasidium spp.;Cephalosporum spp.; Chaetomium spp.; Coprinus spp.; Dactyllum spp.;Fusarium spp., including F. conglomerans, F. decemcellulare, F.javanicum, F. lini, F.oxysporum and F. solani; Gliocladium spp.;Humicola spp., including H. insolens and H. lanuginosa; Mucor spp.;Neurospora spp., including N. crassa and N. sitophila; Nsocallimastixspp.; Orpinomyces spp.; Penicillium spp; Phanerochaete spp.; Phlebiaspp.; Piromyces spp.; Pseudomonas spp.; Rhizopus spp.; Schizophyllumspp.; Trametes spp.; Trichoderma spp., including T. reesei, T. reesei(longibrachiatum) and T. viride; and Zygorhynchus spp. Similarly, it isenvisioned that a swollenin and/or DNA encoding a swollenin as describedherein may be found in cellulolytic bacteria such as Bacillus spp.;Cellulomonas spp.; Clostridium spp.; Myceliophthora spp.;Thermomonospora spp.; Streptomyces spp., including S. olivochromogenes;specifically fiber degrading ruminal bacteria such as Fibrobactersuccinogenes; and in yeast including Candida torresii; C. parapsliosis;C. sake; C. zeylanoides; Pichia minuta; Rhodotorula glutinis; R.mucilaginosa; and Sporobolomyces holsaticus.

[0026] Preferably, swollenin proteins according to the present inventionare isolated or purified. By purification or isolation is meant that theswollenin protein is altered from its natural state by virtue ofseparating the swollenin from some or all of the naturally occurringconstituents with which it is associated in nature. This may beaccomplished by art recognized separation techniques such as ionexchange chromatography, affinity chromatography, hydrophobicseparation, dialysis, protease treatment, ammonium sulphateprecipitation or other protein salt precipitation, centrifugation, sizeexclusion chromatography, filtration, microfiltration, gelelectrophoresis or separation on a gradient to remove whole cells, celldebris, impurities, extraneous proteins, or enzymes undesired in thefinal composition. It is further possible to then add constituents tothe swollenin containing composition which provide additional benefits,for example, activating agents, anti-inhibition agents, desirable ions,compounds to control pH or other enzymes such as cellulase.

[0027] Hybridization is used herein to analyze whether a given fragmentor gene corresponds to the swollenin described herein and thus fallswithin the scope of the present invention. The hybridization assay isessentially as follows: Genomic DNA from a particular target source isfragmented by digestion with a restriction enzyme(s), e.g., EcoR I, HindIII, Bam HI, Cla I, Kpn I, Mlu I, Spe I, Bgl II, Nco I, Xba I, Xho I andXma I (supplied by New England Biolabs, Inc., Beverly, Mass. andBoehringer Mannheim) according to the manufacturer's instructions. Thesamples are then electrophoresed through an agarose gel (such as, forexample, 0.7% agarose) so that separation of DNA fragments can bevisualized by size. The gel may be briefly rinsed in distilled H₂O andsubsequently depurinated in an appropriate solution (such as, forexample, 0.25M HCl) with gentle shaking followed by denaturation for 30minutes (in, for example, 0.4 M NaOH). A renaturation step may beincluded in which the gel is placed in 1.5 M NaCl, IM Tris, pH 7.0 withgentle shaking for 30 minutes. The DNA should then be transferred ontoan appropriate positively charged membrane, for example the MaximumStrength Nytran Plus membrane (Schleicher & Schuell, Keene, N. H.),using a transfer solution (such as, for example, 6×SSC (900 mM NaCl, 90mM trisodium citrate). After the transfer is complete, generally atabout 2 hours or greater, the membrane is rinsed and air dried at roomtemperature after using a rinse solution (such as, for example,2×SSC[2×SSC=300 mM NaCl, 30 mM trisodium citrate]). The DNA should thenbe crosslinked to the membrane by either UV-crosslinking or by baking inan oven using temperatures recommended by the membrane manufacturer. Themembrane should then be prehybridized, (for approximately 2 hours ormore) in a suitable prehybridization solution (such as, for example, anaqueous solution containing per 100 mis: 30-50 mis formamide, 25 mis of20×SSPE (1×SSPE=0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7), 2.5 misof 20% SDS, 1 ml of 10 mg/ml sheared herring sperm DNA).

[0028] A DNA probe taken from the sequence in FIG. 1 should be isolatedby electrophoresis in an agarose gel, the fragment excised from the geland recovered from the excised agarose. This purified fragment of DNA isthen labeled (using, for example, the Megaprime labeling systemaccording to the instructions of the manufacturer to incorporate p³² inthe DNA (Amersham International plc, Buckinghamshire, England)). Thelabeled probe is denatured by heating to 95° C. for 5 minutes andimmediately added to the prehybridization solution above containing themembrane. The hybridization reaction should proceed for an appropriatetime and under appropriate conditions, for example, for 18 hours at 37°C. with gentle shaking. The membrane is rinsed (for example, in2×SSC/0.3% SDS) and then washed with an appropriate wash solution andwith gentle agitation. The stringency desired will be a reflection ofthe conditions under which the membrane (filter) is washed.

[0029] Specifically, the stringency of a given reaction (i.e., thedegree of homology necessary for successful hybridization) will dependon the washing conditions to which the filter from the southern Blot issubjected after hybridization. “Low-stringency” conditions as definedherein will comprise washing a filter from a Southern Blot with asolution of 0.2×SSC/0. 1% SDS at 20° C. for 15 minutes.“Standard-stringency” conditions comprise a further washing stepcomprising washing the filter from the Southern Blot a second time witha solution of 0.2×SSC/0. 1% SDS at 37° C. for 30 minutes.

[0030] “Cellulase” is a well classified category of enzymes in the artand includes enzymes capable of hydrolyzing cellulose polymers toshorter oligomers and/or glucose. Common examples of cellulase enzymesinclude exo-cellobiohydrolases and endoglucanases and are obtainablefrom many species of cellulolytic organisms, particularly includingfungi and bacteria.

[0031] “Hemicellulase” is also a well classified category of enzymes inthe art and includes enzyme capable of hydrolyzing hemicellulosepolymers to shorter oligomers. Common examples of hemicellulases includexylanase and mannanase.

[0032] “Cellulose containing materials” means materials comprisingcellulose polymer as one of its constituents. Cellulose will thusinclude sewn or unsewn fabrics or other articles made of pure cotton orcotton blends including cotton woven fabrics, cotton knits, cottondenims, cotton yarns and the like or blends thereof including one ormore non-cotton fibers including synthetic fibers such as polyamidefibers (for example, nylon 6 and nylon 66), acrylic fibers (for example,polyacrylonitrile fibers), and polyester fibers (for example,polyethylene terephthalate), polyvinyl alcohol fibers (for example,Vinylon), polyvinyl chloride fibers, polyvinylidene chloride fibers,polyurethane fibers, polyurea fibers and aramid fibers. “Cellulose”further means any cotton or non-cotton containing cellulosic fabric orcotton or non-cotton containing cellulose blend including naturalcellulosics and manmade cellulosics (such as jute, flax, ramie, rayon,TENCEL®). Included under the heading of manmade cellulosics areregenerated fabrics that are well known in the art such as rayon. Othermanmade cellulosics include chemically modified cellulose fibers (e.g,cellulose derivatized by acetate) and solvent-spun cellulose fibers. Ofcourse, included within the definition of cellulose containing fabric isany garment or yarn made of such materials. Similarly, “cellulosecontaining fabric” includes textile fibers made of such materials.Additionally, materials comprising cellulose include wood, wood pulp andother plant-based fiber (i.e., grasses, feeds, seeds, trees, cornhusks), paper, cardboard, particle board, nutritional fiber andnon-nutritional fiber.

[0033] “Derivative” means a protein which is derived from a precursorprotein (e.g., the native protein) by addition of one or more aminoacids to either or both the C- and N-terminal end, substitution of oneor more amino acids at one or a number of different sites in the aminoacid sequence, deletion of one or more amino acids at either or bothends of this protein or at one or more sites in the amino acid sequence,or insertion of one or more amino acids at one or more sites in theamino acid sequence. The preparation of a swollenin derivative ispreferably achieved by modifying a DNA sequence which encodes for thenative protein, transformation of that DNA sequence into a suitablehost, and expression of the modified DNA sequence to form the derivativeswollenin. The derivative of the invention includes peptides comprisingaltered amino acid sequences in comparison with a precursor amino acidsequence (e.g., a wild type or native state swollenin), which peptidesretain a characteristic swollenin nature of the precursor swollenin butwhich have altered properties in some specific aspect. For example, aswollenin derivative may have an increased pH optimum or increasedtemperature or oxidative stability but will retain its characteristiccellulose modification activity. Similarly, derivatives according to thepresent invention include a cellulose binding domain which has eitherbeen added, removed or modified in such a way so as to significantlyimpair or enhance its cellulose binding ability. Similarly, a catalyticcellulolytic domain may either be added, removed or modified to operatein conjunction with the swollenin. It is contemplated that derivativesaccording to the present invention may be derived from a DNA fragmentencoding a swollenin derivative wherein the functional activity of theexpressed swollenin derivative is retained. Derivative further includeschemical modification to change the characteristics of the swollenin.

[0034] “Expression vector” means a DNA construct comprising a DNAsequence which is operably linked to a suitable control sequence capableof effecting the expression of the DNA in a suitable host. Such controlsequences may include a promoter to effect transcription, an optionaloperator sequence to control transcription, a sequence encoding suitableribosome-binding sites on the mRNA, and sequences which controltermination of transcription and translation. Different cell types arepreferably used with different expression vectors. A preferred promoterfor vectors used in Bacillus subtilis is the AprE promoter; a preferredpromoter used in E. coli is the Lac promoter, a preferred promoter usedin Saccharomyces cerevisiae is PGK1, a preferred promoter used inAspergillus niger is glaA, and a preferred promoter for Trichodermareesei (longibrachiatum) is cbhl. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may, under suitable conditions, integrate into thegenome itself. In the present specification, plasmid and vector aresometimes used interchangeably. However, the invention is intended toinclude other forms of expression vectors which serve equivalentfunctions and which are, or become, known in the art. Thus, a widevariety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences such as various knownderivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.coli including col E1, pCR1, pBR322, pMb9, pUC 19 and their derivatives,wider host range plasmids, e.g., RP4, phage DNAs e.g., the numerousderivatives of phage λ, e.g., NM989, and other DNA phages, e.g., M13 andfilamentous single stranded DNA phages, yeast plasmids such as the 2μplasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in animal cells and vectors derived from combinationsof plasmids and phage DNAs, such as plasmids which have been modified toemploy phage DNA or other expression control sequences. Expressiontechniques using the expression vectors of the present invention areknown in the art and are described generally in, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press (1989). Often, such expression vectors including theDNA sequences of the invention are transformed into a unicellular hostby direct insertion into the genome of a particular species through anintegration event (see e.g., Bennett & Lasure, More Gene Manipulationsin Fungi, Academic Press, San Diego, pp. 70-76 (1991) and articles citedtherein describing targeted genomic insertion in fungal hosts,incorporated herein by reference).

[0035] “Host strain” or “host cell” means a suitable host for anexpression vector comprising DNA according to the present invention.Host cells useful in the present invention are generally procaryotic oreucaryotic hosts, including any transformable microorganism in whichexpression can be achieved. Specifically, host strains may be Bacillussubtilis, Escherichia coli, Trichoderma reesei (longibrachiatum),Saccharomyces cerevisiae or Aspergillus niger. Host cells aretransformed or transfected with vectors constructed using recombinantDNA techniques. Such transformed host cells are capable of bothreplicating vectors encoding swollenin and its variants (mutants) orexpressing the desired peptide product. In a preferred embodimentaccording to the present invention, “host cell” means both the cells andprotoplasts created from the cells of Trichoderma sp.

[0036] “Signal sequence” means a sequence of amino acids bound to theN-terminal portion of a protein which facilitates the secretion of themature form of the protein outside of the cell. This definition of asignal sequence is a functional one. The mature form of theextracellular protein lacks the signal sequence which is cleaved offduring the secretion process.

[0037] “DNA construct or vector” (used interchangeably herein) means anucleotide sequence which comprises one or more DNA fragments or DNAvariant fragments encoding any of the novel swollenins or derivativesdescribed above.

[0038] “Functionally attached to” means that a regulatory region, suchas a promoter, terminator, secretion signal or enhancer region isattached to a structural gene and controls the expression of that gene.

[0039] Preparation of Swollenin

[0040] The present invention relates to the expression, purificationand/or isolation and use of swollenins and derivatives of swollenins.These swollenins are preferably prepared by recombinant methods.However, swollenin proteins for use in the present invention may beobtained by other art recognized means such as purification from naturalisolates.

[0041] A preferred mode for preparing swollenin according to the presentinvention comprises transforming a Trichoderma sp. host cell with a DNAconstruct comprising at least a fragment of DNA encoding a portion orall of the swollenin functionally attached to a promoter. Thetransformed host cell is then grown under conditions so as to expressthe desired protein. Subsequently, the desired protein product ispurified to substantial homogeneity.

[0042] Preferably, the microorganism to be transformed comprises astrain derived from Trichoderma spp. or Aspergillus spp. Morepreferably, the strain comprises T. reesei (longibrachiatum) which isuseful for obtaining overexpressed protein or Aspergillus niger var.Awamori. For example, RL-P37, described by Sheir-Neiss et al. in Appl.Microbiol. Biotechnology, 20 (1984) pp. 46-53 is known to secreteelevated amounts of cellulase enzymes. Functional equivalents of RL-P37include Trichoderma reesei (longibrachiatum) strain RUT-C30 (ATCC No.56765) and strain QM9414 (ATCC No. 26921). Another example includesoverproducing mutants as described in Ward et al. in Appl. Microbiol.Biotechnology 39:738-743 (1993). It is contemplated that these sitrainswould also be useful in overexpressing Trichoderm spp. swollenin.

[0043] Where it is desired to obtain the swollenin protein in theabsence of cellulolytic activity, it is useful to obtain, for example, aTrichoderma host cell strain which has had one or more cellulase genesdeleted prior to introduction of a DNA construct or plasmid containingthe DNA fragment encoding the swollenin. Such strains may be prepared bythe method disclosed in U.S. Pat. No. 5,246,853 and WO 92/06209, whichdisclosures are hereby incorporated by reference. By expressing aswollenin in a host microorganism that is missing one or more cellulasegenes, the identification and subsequent purification procedures aresimplified. Any gene from Trichoderma sp. which has been cloned can bedeleted, for example, the cbh1, cbh2, egl1, and egl3 genes as well asthose encoding EGIII and/or EGV protein (see e.g., U.S. Pat. No.5,475,101 and WO 94/28117, respectively).

[0044] Gene deletion may be accomplished by inserting a form of thedesired gene to be deleted or disrupted into a plasmid by methods knownin the art. The deletion plasmid is then cut at an appropriaterestriction enzyme site(s), internal to the desired gene coding region,and the gene coding sequence or part thereof replaced with a selectablemarker. Flanking DNA sequences from the locus of the gene to be deletedor disrupted, preferably between about 0.5 to 2.0 kb, remain on eitherside of the selectable marker gene. An appropriate deletion plasmid willgenerally have unique restriction enzyme sites present therein to enablethe fragment containing the deleted gene, including flanking DNAsequences, and the selectable marker gene to be removed as a singlelinear piece.

[0045] A selectable marker must be chosen so as to enable detection ofthe transformed fungus. Any selectable marker gene which is expressed inthe selected microorganism will be suitable. For example, withTrichoderma sp., the selectable marker is chosen so that the presence ofthe selectable marker in the transformants will not significantly affectl:he properties thereof. Such a selectable marker may be a gene whichencodes an assayable product. For example, a functional copy of aTrichoderma sp. gene may be used which if lacking in the host strainresults in the host strain displaying an auxotrophic phenotype.

[0046] In a preferred embodiment, a pyr4⁻ derivative strain ofTrichoderma sp. is transformed with a functional pyr4 gene, which thusprovides a selectable marker for transformation. A pyr4⁻ derivativestrain may be obtained by selection of Trichoderma sp. strains which areresistant to fluoroorotic acid (FOA). The pyr4 gene encodesorotidine-5′-monophosphate decarboxylase, an enzyme required for thebiosynthesis of uridine. Strains with an intact pyr4 gene grow in amedium lacking uridine but are sensitive to fluoroorotic acid. It ispossible to select pyr4⁻ derivative strains which lack a functionalorotidine monophosphate decarboxylase enzyme and require uridine forgrowth by selecting for FOA resistance. Using the FOA selectiontechnique it is also possible to obtain uridine requiring strains whichlack a functional orotate pyrophosphoribosyl transferase. It is possibleto transform these cells with a functional copy of the gene encodingthis enzyme (Berges and Barreau, 1991, Curr. Genet. 19 pp. 359-365).Selection of derivative strains is easily performed using the FOAresistance technique referred to above, and thus, the pyr4 gene ispreferably employed as a selectable marker.

[0047] To transform pyr4⁻ Trichoderma sp. so as to be lacking in theability to express one or more cellulase genes, a single DNA fragmentcomprising a disrupted or deleted cellulase gene is then isolated fromthe deletion plasmid and used to transform an appropriate pyrTrichoderma host. Transformants are then identified and selected basedon their ability to express the pyr4 gene product and thus complimentthe uridine auxotrophy of the host strain. Southern blot analysis isthen carried out on the resultart transformants to identify and confirma double crossover integration event which replaces part or all of thecoding region of the genomic copy of the gene to be deleted with thepyr4 selectable markers.

[0048] Although the specific plasmid vectors described above relate topreparation of pyr transformants, the present invention is not limitedto these vectors. Various genes can be deleted and replaced in theTrichoderma sp. strain using the above techniques. In addition, anyavailable selectable markers can be used, as discussed above. In fact,any Trichoderma sp. gene which has been cloned, and thus identified, canbe deleted from the genome using the above-described strategy.

[0049] As stated above, the host strains used are derivatives ofTrichoderma sp. which lack or have a nonfunctional gene or genescorresponding to the selectable marker chosen. For example, if theselectable marker of pyr4 is chosen, then a specific pyr4⁻ derivativestrain is used as a recipient in the transformation procedure.Similarly, selectable markers comprising Trichoderma sp. genesequivalent to the Aspergillus nidulans genes, amdS, argB, trpC, niaD maybe used. The corresponding recipient strain must therefore be aderivative strain such as argB⁻, trpC⁻, niaD⁻, respectively.

[0050] DNA encoding the swollenin protein is then prepared for insertioninto an appropriate microorganism. According to the present invention,DNA encoding for a swollenin enzyme comprises all of the DNA necessaryto encode for a protein which has functional swollenin activity.Accordingly, DNA may be derived from any microbial source which producesswollenin, provided that the gene may be identified and isolatedpursuant to the methods described herein. In a preferred embodiment, theDNA encodes for an swollenin protein derived from Trichoderma sp., andmore preferably from Trichoderma reesei (longibrachiatum).

[0051] The DNA fragment: or DNA variant fragment encoding the swolleninor derivative may be functionally attached to a fungal promotersequence, for example, the promoter of the cbh1 or egl1 gene.

[0052] It is also contemplated that more than one copy of DNA encoding aswollenin may be recombined into the strain to facilitateoverexpression.

[0053] The DNA encoding the swollenin may be prepared by theconstruction of an expression vector carrying the DNA encoding thetruncated cellulase. The expression vector carrying the inserted DNAfragment encoding the swollenin may be any vector which is capable ofreplicating autonomously in a given host organism or of integrating intothe DNA of the host, typically a plasmid. In preferred embodiments twotypes of expression vectors for obtaining expression of genes arecontemplated. The first contains DNA sequences in which the promoter,gene coding region, and terminator sequence all originate from the geneto be expressed. Gene truncation may be obtained by deleting awayundesired DNA sequences (e.g., coding for unwanted domains) to leave thedomain to be expressed under control of its own transcriptional andtranslational regulatory sequences. A selectable marker is alsocontained on the vector allowing the selection for integration into thehost of multiple copies of the novel gene sequences.

[0054] The second type of expression vector is preassembled and containssequences required for high level transcription and a selectable marker.It is contemplated that the coding region for a gene or part thereof canbe inserted into this general purpose expression vector such that it isunder the transcriptional control of the expression cassettes promoterand terminator sequences. For example, pTEX is such a general purposeexpression vector. Genes or part thereof can be inserted downstream ofthe strong cbh1 promoter.

[0055] In the vector, the DNA sequence encoding the swollenin of thepresent invention should be operably linked to transcriptional andtranslational sequences, i.e., a suitable promoter sequence and. signalsequence in reading frame to the structural gene. The promoter may beany DNA sequence which shows transcriptional activity in the host celland may be derived from genes encoding proteins either homologous orheterologous to the host cell. The signal peptide provides forextracellular production of the swollenin or derivatives thereof. TheDNA encoding the signal sequence is preferably that which is naturallyassociated with the gene to be expressed, however the signal sequencefrom any suitable source, for example an exo-cellobiohydrolases orendoglucanase from Trichoderma, is contemplated in the presentinvention.

[0056] The procedures used to ligate the DNA sequences coding for theswollenins of the present invention with the promoter, and insertioninto suitable vectors are well known in the art.

[0057] The DNA vector or construct described above may be introduced inthe host cell in accordance with known techniques such astransformation, transfection, microinjection, microporaton, biolisticbombardment and the like.

[0058] In the preferred transformation technique, it must be taken intoaccount that the permeability of the cell wall to DNA in Trichoderma sp.is very low. Accordingly, uptake of the desired DNA sequence, gene orgene fragment is at best minimal. There are a number of methods toincrease the permeability of the Trichoderma sp. cell wall in thederivative strain (i.e., lacking a functional gene corresponding to theused selectable marker) prior to the transformation process.

[0059] The preferred method in the present invention to prepareTrichoderma sp. for transformation involves the preparation ofprotoplasts from fungal mycelium. The mycelium can be obtained fromgerminated vegetative spores. The mycelium is treated with an enzymewhich digests the cell wall resulting in protoplasts. The protoplastsare then protected by the presence of an osmotic stabilizer in thesuspending medium. These stabilizers include sorbitol, mannitol,potassium chloride, magnesium sulfate and the like. Usually theconcentration of these stabilizers varies between 0.8 M to 1.2 M. It ispreferable to use about a 1.2 M solution of sorbitol in the suspensionmedium.

[0060] Uptake of the DNA into the host Trichoderma sp. strain isdependent upon the calcium ion concentration. Generally between about 10mM CaCl₂ and 50 mM CaCl₂ is used in an uptake solution. Besides the needfor the calcium ion in the uptake solution, other items generallyincluded are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulforiic acid)and polyethylene glycol (PEG). It is believed that the polyethyleneglycol acts to fuse the cell membranes thus permitting the contents ofthe medium to be delivered into the cytoplasm of the Trichoderma sp.strain and the plasmid DNA is transferred to the nucleus. This fusionfrequently leaves multiple copies of the plasmid DNA tandemly integratedinto the host chromosome.

[0061] Usually a suspension containing the Trichoderna sp. protoplastsor cells that have been subjected to a permeability treatment at adensity of 10⁸ to 10⁹/ml, preferably 2×10⁸/ml are used intransformation. A volume of 100 microliters of these protoplasts orcells in an appropriate solution (e.g., 1.2 M sorbitoi; 50 mM CaCl₂) aremixed with the desired DNA. Generally a high concentration of PEG isadded to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 canbe added to the protoplast suspension. However, it is preferable to addabout 0.25 volumes to the protoplast suspension. Additives such asdimethyl sulfoxide, heparin, spermidine, potassium chloride and the likemay also be added to the uptake solution and aid in transformation.

[0062] Generally, the mixture is then incubated at approximately 0° C.for a period of between 10 to 30 minutes. Additional PEG is then addedto the mixture to further enhance the uptake of the desired gene or DNAsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is preferably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then incubated at room temperature beforethe addition of a sorbitol aid CaCl₂ solution. The protoplast suspensionis then further added to molten aliquots of a growth medium. This growthmedium permits the growth of transformants only. Any growth medium canbe used in the present invention that is suitable to grow the desiredtransformants. However, if Pyr⁺ transformants are being selected it ispreferable to use a growth medium that contains no uridine. Thesubsequent colonies are transferred and purified on a growth mediumdepleted of uridine.

[0063] At this stage, stable transformants may be distinguished fromunstable transformants by their faster growth rate and the formation ofcircular colonies with a smooth, rather than ragged outline on solidculture medium lacking uridine. Additionally, in some cases a furthertest of stability may made by growing the transformants on solidnon-selective medium (i.e. containing uridine), harvesting spores fromthis culture medium and determining the percentage of these spores whichwill subsequently germinate and grow on selective medium lackinguridine.

[0064] In a particular embodiment of the above method, the swollenins orderivatives thereof are recovered in active form from the host cellafter growth in liquid media either as a result of the appropriate posttranslational processing of the novel swollenin or derivatives thereof.

[0065] The expressed swollenins are recovered from the medium byconventional techniques including separations of the cells from themedium by centrifugation, filtration, and precipitation of the proteinsin the supernatant or filtrate with a salt, for example, ammoniumsulphate. Additionally, chromatography procedures such as ion exchangechromatography or affinity chromatography may be used. Antibodies(polyclonal or monoclonal may be raised against the natural purifiedswollenins, or synthetic peptides may be prepared from portions of theswollenin molecule and used to raise polyclonal antibodies.

EXAMPLE 1

[0066]Trichoderma reesei (longibrachiatum) cDNA Clone Encoding a NovelSwollenin

[0067]FIG. 1 shows the nucleotide sequence (SEQ ID:NO 1) and predictedcorresponding amino acid sequence (SEQ ID:NO 2) of a cDNA clone obtainedfrom a library of cDNA prepared from Trichoderma reesei(longibrachiatum) RNA after growth on a mixed carbon source as describedby Saloheimo et al. 1994, Molec. Microbiol. 13:219-228. The cDNA showedthe following characteristics which help to describe the gene:

[0068] An open reading frame of 1482 nt was identified and the encodedprotein was deduced.

[0069] The first 18 amino acids of the predicted protein have thefollowing features expected of a secretion signal sequence and signalcleavage site. There is a positively charged amino acid (lysine) closeto the amino-terminal methionine which is followed by a sequence ofhydrophobic amino acids and an apparent signal peptidase cleavage sitefollowing amino acid lle18. The predicted N-terminus of the matureswollenin would therefore be Gln-Gln. Similarly, many of the maturecellulases produced by Trichoderma have glutamine at the N-terminus(e.g., CBHI, CBHII, EGI, EGII and EGIII) and both EGI and EGII beginwith a pair of glutamine residues reinforcing the conclusion that thisis the N-terminus. The mature protein is therefore predicted to be 475amino acids in length and have a molecular weight of approximately 49.5kDa, riot including any possible glycosylation or other modification,and a calculated pl of approximately 4.6 based on the amino acidcomposition. There are three potential N-linked glycosylation sites(having the consensus amino acid sequence of N-X-S/T) at Asparagines160, 336 and 406.

[0070] Residues 4 to 39 of the predicted mature protein sequence haveclose similarity with the cellulose binding domains (CBDs) of cellulasesproduced by Trichoderma and other fungal cellulases (58% identity withthe CBD of CBHII of Trichoderma). CBDs are also associated with somenon-cellulolytic extracellular fungal enzymes such as acetyl xylanesterase and mannanase from Trichoderma reesei (longibrachiatum) andsimilar identity is shown between swollenin CBD and these CBD's.

[0071] Following the CBD of the predicted Trichoderma protein is aregion (from residue 41 to approximately residue 86) which is rich inSer, Thr, Gly and Pro residues and which should share a similarfunctionality to the linker or hinge regions present in Trichoderma andother fungal cellulases and which connect the CBD with the catalyticdomain.

[0072] Regions of similarily are observed between the predicted aminoacid sequence (SEQ ID NO: 2) of the Trichoderma swollenin of FIG. 1 andknown sequences of higher plant expansins. FIG. 2 shows an alignmentbetween part of the predicted Trichoderma protein and a consensussequence (SEQ ID NO: 3) derived from nine plant expansins by Shcherbanet al., supra. These sequences were aligned using the Jotun Heinalgorithm within the Lasergene software package (DNASTAR Inc.) and a 36%similarity was calculated between the two amino acid sequences. Of the322 amino acids of Trichoderma swollenin sequence used in thisalignment, 70 or 21.7% are identical to the higher plant consensussequence.

[0073] Regions of similarity can also be observed between theTrichoderma reesei (longibrachiatum) swollenin and human titin proteinthat is rich in fibronectin type repeats. The homology was detected in asimilarity search to the protein sequence databanks carried out with theprogram BLAST (Altschul et al., 1990, J. Mol. Biol. 215:403-410) and thealignments shown as examples have been created by the program. Theregions of titin homologous to the T. reesei swollenin are parts of thefibronectin type repeats. Fibronectin repeats have been found in somebacterial carbohydrate-modifying enzymes (Little et al., 1994, J. Mol.Evol. 39:631-643) but not from any fungal protein. A BLAST searchreveals no similarity between the plant expansins and fibronectin repeatcontaining proteins. T.r. swo 283 GGPYYFALTAVNTNGPGSVTKI (SEQ. ID NO:21) Human titin 12268 GNEYYFRVTAVNEYGPGVPTDV (SEQ. ID NO: 22) T.r. swo100 TKGSVTASWTDPMETLGA (SEQ. ID NO: 23) Human titin 9114TKGSMLVSWTPPLDNGGS (SEQ. ID NO: 24)

[0074] The Trichoderma reesei (longibrachiatum) swollenin gene wasexpressed when the fungus was grown on cellulose as the sole carbonsource, but not when grown on glucose as the sole carbon source.

[0075] In order to investigate the regulation of swollenin geneexpression in Trichoderma the following experiment was performed.Trichoderma reesei (longibrachiatum) strain QM9414 was grown in shakeflasks (28° C., 200 RPM) in a minimal medium (Penttilä et al., 1987,Gene 61:155-164) containing 5% glucose or 2% cellulose for three days.To test for sophorose induction, the strain was grown in a minimalmedium with 20%, sorbitol for three days and sophorose was added to thefinal concentration of 1 mM. The culture was continued for another tenhours and the same amount of sophorose was added. The cultivation wasended five hours after the second addition. A 87 h cultivation in 2%sorbitol was carried out without sophorose additions as a control. Afterthe cultivations the mycelium was harvested by filtration with a glassfibre filter, washed with 0.9% NaCl and frozen. Total RNA was isolatedfrom the mycelial samples according to Chirgwin et al. (1979, Biochem.J. 18:5294-5299). RNA samples of 5 μg were treated with glyoxal and runin a 1% agarose gel in 10 mM Na-phosphate buffer, pH 7. Capillaryblotting onto a Hybond-N nylon membrane (Amersham) was carried outaccording to manufacturer's instructions. The hybridization probe wasprepared by digesting the cDNA library plasmid carrying the swollenincDNA with EcoRI and XhoI, running the digested plasmid in a 0.8%agarose! gel and isolating the cDNA fragment from the gel with theQiaquick gel extraction kit (Qiagen). The probe was labelled with³²P-dCTP using the Random Primed DNA labelling kit (BoehringerMannheim). Hybridization was one for 24h at 42° C. in 50% formamide, 10%dextran sulphate, 1% SDS, 1M NaCL, 125 μg/ml herring sperm DNA. Thefilter was washed at 42° C. in 5×SSPE for 15 minutes, in 1×SSPE, 0.1%SDS for 2×15 minutes and in 0.1×SSPE, 0.1% SDS 2×15 minutes at roomtemperature. (1×SSPE is 0.18 M NaCL, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7).The results of this experiment are shown in FIG. 3. No swollenin mRNAwas observed after growth on glucose and very little was observed aftergrowth on sorbitol. In contrast, high levels of swollenin mRNA wereobserved after growth on cellulose or after addition of sophorose to asorbitol-grown culture.

EXAMPLE 2 Preparation of a Cloned DNA Molecule Encoding TrichodermaSwollenin

[0076] The following is provided as a method of preparing a clonecomprising an entire swollenin gene described in Example 2. In thisexample, genomic DNA or cDNA clones derived from Trichoderma and areprepared by using the following procedure.

[0077] The oligonucleotides shown below are synthesized: EXP-A5′-GGCGAGATCTTGCTGCCCATCATATTGTGC-3′ (SEQ ID NO:19) EXP-B5′-GGCGTCTAGACTGCACACCAATGTCAATGT-3′ (SEQ ID NO:20)

[0078] Oligonucleotide EXP-A contains a BgIII restriction enzymerecognition site near the 5′ end followed by the DNA sequence from nt425 to nt 445 of SEQ ID NO:1. Oligonucleotide EXP-B contains an Xbalrecognition site near the 5′ end followed by the reverse complement ofthe DNA sequence from nt 1471 to nt 1490 of SEQ ID NO:1.

[0079] Polymerase chain reaction (PCR) was performed using theoligonucleotides EXP-A and EXP-B as primers and total genomic DNAisolated from Trichoderma reesei strain QM6a (ATCC; 13631) as template.The DNA polymerase enzyme (Pwo polymerase), buffer and deoxynucleotidemixture used were supplied by Boehringer Mannheim. The followingconditions were used for PCR; step 1, 1 min. at 94° C.; step 2, 40 sec.at 92° C.; step 3, 1 min. at 50° C., step 4, 2 min. at 72° C.; steps 2,3 and 4 repeated 29 times; step 5, 5 min. at 72° C.

[0080] The major DNA product of PCR was a fragment of approximately 1.3kb as estimated by agarose gel electrophoresis. The PCR product wasdigested with Bg/II and XbaI and the 1.3 kb DNA fragment was purifiedfrom an agarose electrophoresis gel. This DNA fragment was ligated withpSL 1180 (Pharmacia) which had been digested with Bg/II and XtaI. Theresulting plasmid was named pSLexpPCR. DNA sequence analysis confirmedthat the 1.3 kb insert in pSLexpPCR corresponded to the expectedfragment of the Trichoderma swollenin gene. The DNA sequence revealedthe presence of three introns within this 1.3 kb fragment at positionscorresponding to between nt 575 and nt 576, between nt 791 and nt 792,and between nt 969 and nt 970 of SEQ ID NO:1.

[0081] The plasmid, or the 1.3 kb insert it contains, can now be used asa hybridization probe to allow the entire swollenin gene to be clonedfrom any genomic DNA or cDNA libraries of interest. The swolleninencoding DNA within the pSLexpPCR does not included the regionscorresponding to the CBD or the linker (hinge) region. Therefore. bydesign, it would be expected to hybridize with other swollenin DNAsequences but not to CBD encoding sequences which may be part of othernon-swollenin genes.

[0082] Total genomic DNA from T. reesei (longibrachiatum) strain QM6awas digested separately with a variety of different restrictionendonucleases and subjected to agarose gel electrophoresis. The DNA wassubsequently blotted to a Nytran (S&S) membrane filter and probed withthe 1.3 kb Bg/II-XbaI DNA fragment isolated from pSLexpPCR and labeledwith ³²P by the Megaprime random labeling system supplied by Amersham.Hybridization with the probe was performed at moderate stringency in abuffer containing 30% formamide, 5×SSPE, 0.5% SDS at 38° C. The membranefilter was subsequently washed at moderate stringency in 2×SSC, 0.1% SDSat 55° C. before being exposed to X-ray film. The results indicated thatthe genomic copy of the T. reesei swollenin gene resides on anapproximately 4.5 kb BgIII fragment, or on an approximately 5.5 kb XbaIfragment.

[0083] Given the exemplified swollenin gene as provided above, it wouldbe routine for one of skill in the art to clone the Trichoderma reeseiswollenin gene from genomic DNA or cDNA libraries by colonyhybridization using the PCR fragment inserted in pSLexpPCR as a probe.

EXAMPLE 3 Cloning the Genomic Copy of T. Reesei Swollenin and Expressionof it in Aspergillus nicer var. awamori

[0084] The genomic copy of T. reesei swollenin was cloned by PCR. Thetemplate DNA was from T. reesei RutC-30 (ATCC 56765) and the primerscorresponding to the 5′ and 3′ ends of the swollenin coding region weredesignated as GCI-PVS-055 (gcg cag atc tca gca atg cjct ggt aag ctt atcctc g) and GCI-PVS-056 (gcg ctc tag atc aat tct ggc taa act gca cac c).

[0085] The PCR-amplified fragment was digested with Bg/II and XbaI andcloned into a Bg/II-XbaI opened pGAPT-PT resulting in pGAPT-expC.Sequencing the insert revealed that the chromosomal copy of theswollenin gene has five introns.

[0086] The chromosomal copy of the swollenin gene (i.e. pGAPT-expC) wastransformed into Aspergillus and transformants were screened asdescribed above for the cDNA.

EXAMPLE 4 Method of Isolating DNA Sequences Encoding Swollenins inMicroorganisms

[0087] The general technique in Examples 2 and 3 may be adapted inconjunction with known techniques to obtain clones comprising swolleninor swollenin-type genes from other fungi and bacteria. Plasmid pSLexpPCRor the isolated 1.3 kb DNA insert encoding part of the swollenin gene(Example 2), may be labelled as can the core region of the swollenin(Example 3). This DNA probe can then be used to hybridize with genomicDNA or cDNA from other fungi or bacteria. Sequences which have beenpublished for higher plant expansins show a very high level of aminoacid identity (see, e.g., FIG. 4, where underlined segments indicateregions of high homology). A comparison of the deduced amino acidsequence of the Trichoderma swollenin with the known amino acidsequences of higher plant expansins identifies certain conserved regionsof amino acids between the swollenins and plant expansins. Theseconserved regions provide the basis for designing degenerate primers foruse in PCR amplification of swollenin-encoding DNA from othermicroorganisms. Such methods are generally known in the art andconsidered routine (see e.g., McPherson et al., PCR A PracticalApproach, pp. 171-186 (1991)). Conserved regions corresponding to aminoacids 192-200 and 366-371 of SEQ ID NO:2 are pointed to as beingparticularly useful for this purpose (see also, highlighted segments ofFIG. 2 although other conserved regions could be used.

[0088] The sequence at amino acid residues 192-200 of SEQ ID NO:2,TSGGACGFG (SEQ. ID NO:14), is highly homologous to the correspondingsequence in the consensus plant expansin sequence TMGGACGYG (SEQ. ID NO.15)(numbered positions 19-27 in FIG. 4). Based on this region ofhomology, it would be possible to synthesize degenerate oligonucleotidescomprising all possible DNA sequences which encode part or all of theamino acid sequence T(M/S)GGACG(Y/F)G (see e.g., McPherson et al.,supra, page 174).

[0089] The sequence at amino acid residues 366 to 371 of SEQ ID:NO.2,YRRVQC (SEQ. ID NO. 16), is highly homologous to the correspondingsequence in the consensus plant expansin sequences YRRVPC (SEQ ID.NO:17) and FRRVPC (SEQ. ID NO: 18) (numbered positions 127-132 in FIG.4). Based on this region of homology, it would also be possible tosynthesize degenerate oligonucleotides to include all possible DNAsequences which encode part or all of the amino acid sequence(F/Y)RRV(P/Q)C. The oligonucleotides derived from this amino acidsequence would be used in conjunction with those derived from theprevious mentioned amino acid sequence as primers for routine PCRexperiments using genomic DNA. Genomic DNA or cDNA could then easily beobtained from any microbe and used as a template in such PCRexperiments. In this way it would be possible to clone genes encodingswollenins from a variety of microbes.

EXAMPLE 5 Heterologous Hybridization Method for Isolating SwolleninEncoding Sequences from Other Microorganisms

[0090] Genomic DNA from different microorganisms was digested with Hind3and run on 1.0% agarose gel. Gel was depurinated, denatured and blotted,and the membrane was UV-crosslinked as described on page 6.Prehybridization, hybridization, labeling of the probe and detectionwere done using the DIG/Genius™ System from Boehringer Mannheim.

[0091] The probe corresponded to the sequence encoding the core regionof T. reesei swollenin. The original cDNA subclone (EXAMPLE 1) wasdigested with Nco1 and EcoR1 resulting in a 312 bp DNA fragment whichwas labeled with DIG-dUTP (dioxigenin-dUTP) via random-primed labelingaccording to manufacturer's (Boehringer Mannheim) instructions.

[0092] The membrane was prehybridized and hybridized in 5×SSC-0.1%N-lauroylsarcosine-0.02% SDS-1% Genius™ blocking Hybridization (overnight) was followed by two 10 minute washes in 6×SSC at room temperatureand two 5 minute washes in 6×SSC at 45° C. Detection with ananti-DIG-alkaline phosphatase conjugate and visualization with achemiluminescence substrate CSPD® were done according to manufacturer'sinstructions.

[0093] Results from this experiment indicated that at least thefollowing species, in addition to T. reesei, hybridize to the probe:Trichoderma koningii, Hypocrea lenta and Hypocrea schweinitzii. In thisHind3 digestion T. reesei and T. koningii had a over 5 kb band thathybridized with the T reesei swollenin gene. For H. schweinitzii, theband that hybridized was 3.7 kb and for H. lenta approximately 3.3 kb insize. This method and variations of it (different hybridization andwashing conditions) can be used to detect swallenin encoding genes fromany organisms.

EXAMPLE 6 Preparation Of A Saccharomyces cerevisiae Clone for Expressionof T. reesei Swollenin

[0094] During the course of obtaining the Trichoderma reesei cDNAmentioned in Example 1, a Saccharomyces cerevisiae clone was obtainedwhich contained an expression plasmid in which the cDNA sequence of SEQID NO:1 was inserted between the S. cerevisiae PGK1 promoter and theterminator region in plasmid pAJ401 (Saloheimo et al., 1994, Molec.Microbiol., Vol. 13, pp. 219-228 (1994)) according to the methoddescribed by Margolles-Clark et al., (Appl. Environ. Microbiol.,62:3840-3846, 1996). Briefly, T. reesei cDNA was ligated to theEcoRI-XhoI cut plasmid pAJ401. Plasmid pAJ401 was derived from plasmidpFL60 (Minet and Lacroute, Curr. Genet., Vol. 18, pp. 287-291 (1990) bychanging the two cloning sites EcoRI and XhoI between the yeast PGKpromoter and terminator into the reverse orientation using specificlinkers. Transformation of E. coli strain JS4 by electroporation(Bio-Rad) according to the manufacturer's instructions yields a libraryof 1.3×10⁶ independent clones. One of these clones contained pAJ401 withthe cDNA of SEQ ID NO:1 inserted between the EcoRI and XhoI sites andwas subsequently transformed into S. cerevisiae strain DBY746. A secondyeast clone was obtained which contained pAJ401 without the cDNAsequence of SEQ ID NO:1 for use as a control in Examples 5 and 6.

[0095] The two yeast clones, one control clone and one clone containingthe T. reesei (longibrachiatum) swollenin cDNA sequence, were culturedfor 2-3 days in fermentors. Either Chemap CMF mini 1 liter or Biolafitte14 L fermentors were used. The culture medium was synthetic completemedium without uracil (Sherman, 1991, Methods Enzymol. 194, 3-21). pHwas maintained at 5.0, aeration rate was 1 L/min for the smallerfermentors and 8 L/min for the larger fermentors, and agitation speedwas 300-600 rpm. Following fermentation, the cells were removed bycentrifugation and the supernatant was concentrated 50-100 fold.

EXAMPLE 7 Expression of T. reesei Swollenin cDNA in Aspergillus nigervar. awamori

[0096] Construction of the Aspergillus Expression Vector

[0097] Construction of the Aspergillus expression vector for expressionof T. reesei swollenin cDNA consisted of three steps: (1)PCR-amplification of the swollenin cDNA and subcloning it intopSP73-hind3 (i.e. HindIII site was killed), (2) exchanging the middlepart of the PCR-derived swollenin gene to the original swollenin genefrom the cDNA subclone in order to eliminate mistakes derived fromPCR-amplification, and (3) subcloning the swollenin-insert into aAspergillus expression vector pGAPT-PT for expression under the A. nigervar. awamori glaA promoter (glucoamylase).

[0098] 1. PCR-amplification of the Swollenin cDNA

[0099] Primers ExAspBgl2 (CATTGATCTCAGCMTGGCTGGTMGCTTATCCTC) andExAspXba1 (CGACTCTAGGATTAGTTCTGGCTAAACTGCACACC) were used forPCR-amplification of the coding region of the T. reesei swollenin cDNA(vector from example 1).

[0100] ExAspBgl2 has a Bg/II cloning site which is followed by the fivelast nucleotides of the glaA (glucoamylase) promoter sequence whichprecede the translation start site (ATG). The ATG in ExAspBgl2 isfollowed by a 19-mer corresponding to the swollenin signal sequence.ExAspXba1 has a XbaI cloning site, a STOP codon and a sequence whichcodes for the last 7 codons of the swollenin gene.

[0101] The PCR-amplified 1.5 kb swollenin fragment was digested withBg/II and XbaI and ligated into Bg/II-XbaI opened pSP73-Hind3 vector.Before this cloning step pSP73 (Promega) was first deleted for itsHindIII site. This was done by opening the vector (pSP73) with HindIIIand the protruding ends were filled in with T4 polymerase (with dNTPs),before ligating the vector back together. This vector was designated aspSP73-Hind3.

[0102] pSP73-Hind3 containing the 1.5 kb swollenin insert was designatedas pPCRAexp.

[0103] 2. Replacing the PCR-amplified Sequence with the OriginalSequence

[0104] pPCRAexp was digested with HindIII and BstEII. HindIII cuts theswollenin coding sequence within the signal sequence and BstEIl is closeto the end of the swollenin coding sequence. The 1.4 kb HindIII-BstEIIswollenin fragment from pPCRAexp was discarded and replaced with the 1.4kb HindIII-BstEII swollenin fragment from the original swollenin cDNAsubclone (EXAMPLE 1). The resultant vector was designated as pWTAexp.

[0105] 3. Cloning into the Expression Vector

[0106] pWTrAexp was digested with Bg/II and XbaI resulting in a 1.5 kbswollenin insert with a complete coding region preceded by fivenucleotides of the glaA promoter sequence and flanked by cloning sitesenabling ligation between the glaA promoter and terminator sequences ina Aspergillus expression vector pGAPT-PG (described below). The insertand vector sequences were ligated and the resultant vector wasdesignated as pGAPT-exp (6.5 kb). This is the vector for expressing T.reesei swollenin cDNA in A. niger.

[0107] The expression vector pGAPT-PG (5.1 kb) used for construction ofpGAPT-exp consists of a 1.1 kb Spel-Bg/II fragment of A. niger var.awamori glaA promoter sequence, 0.2 kb fragment of A. niger glaAterminator sequence and 1.6 kb A. nidulans pyrG marker gene in pUC18backbone. The glaA terminator fragment follows the glaA promotersequence and is separated from it by multiple cloning sites which can beused for inserting sequences to be expressed.

[0108] The 3′ end of the glaA promoter sequence, i.e. the sequencepreceding the translation start site of the swollenin gene in pGAPT-exphas been engineered (multiple cloning sites) and has the followingsequence starting from a Xmnl site in the glaA promoter:GAAGTGCTTCCTCCCTTTTAGACGCAACTGAGAGCCTGAGCTTCATCCCCAGCATCATTAGATCTCAGCAATG

[0109] in which the ATG in the end is the start codon for the swollenincDNA.

[0110] The surrounding sequence of the STOP codon is following (startingfrom the ‘TAA’ stop codon—engineered from the original ‘TGA’ STOP codonin swollenin): TAATCCTTCTAGAGTCGACCGCGACGGTGACC

[0111] shown up till the BstEII site (GGTGACC) in the glaA terminatorsequence.

[0112] Transformation of pGAPT-exp to Aspergillus

[0113] pGAPT-exp was transferred to the strain A. niger var. awamoridgr246 p2 described in Ward et al. Appl. Microbiol. Biotechnol.39:738-743 (1993). Transformation of Aspergillus follows the same basicprocedure as described for Trichoderma on pages 13-15. Thetransformation procedure of A. niger var. awamori dgr246 p2 is alsodescribed in Ward et al. Appl. Microbiol. Biotechnol. 39:738-743 (1993).

[0114] Transformants were selected on their ability to grow on minimalnutrients without uridine. The untransformed cells require uridine forgrowth.

[0115] Screening of Transformants

[0116] Aspergillus transformants were cultivated in 50 ml liquid mediumin 250 ml shake flasks for 5-11 days as described in Ward et al.Bio/Technology 8:435-440 (1990). The complex medium contained 15%maltose to induce the glaA promoter and therefore drive expression ofthe swollenin gene. Culture supernatants were run on SDS-PAGE gels.Aspergillus transformants which were producing the T. reesei swolleninhad a band running above the 66 kD marker band and this band was missingfrom lanes of the negative control (Aspergillus strain before thetransformation) (FIG. 6).

EXAMPLE 8 Effect of Treatment with Trichoderma reesei Swollenin onCellulose Structure

[0117] Whatman No. 3 filter paper circles were cut into strips measuring2×7 cm. Buffer used was 50 mM sodium acetate, pH 5. The filter paperstrips were soaked for at least 30 min. at room temperature in solutionsconsisting of water, buffer, 8M urea in buffer, or broth produced fromyeast cones containing the T. reesei swollenin gene or a control yeastclone which does not produce T. reesei swollenin in buffer (dilutionsranged from 1 ml of broth in 7 ml buffer to 4 ml broth in 4 ml buffer).

[0118] A Thwing-Albert tensile tester was set for a test speed of 0.10cm/min and tensile energy measured over a range of 0 to 50 lbs. Eachstrip of filter paper was placed between the clamps and the peak loadwas measured. The results of this experiment quantify the degree of loadthat can be held before breaking the paper. Two or three strips weremeasured for each sample type. The results from several differentexperiments are given below in Tables 1 and 2. TABLE I Sample Trial 1Trial 2 Trial 3 Average buffer .55 .58 .59 .57 8M urea N/A .36 .32 .34control broth .49 .49 .47 .48 swo broth .40 .42 .42 .41

[0119] TABLE 2 Sample Trial 4 Trial 5 Average buffer .56 .59 .58 8M urea.42 .41 .42 control 1 ml .52 .52 .52 control 3 ml .52 .47 .50 swo 1 ml.43 .42 .43 swo 3 ml .46 .40 .43

[0120] As expected, the strips treated with 8M urea, which is known todisrupt hydrogen bonding interactions, cannot hold as high of a loadwithout breaking as strips treated with buffer only. In bothexperiments, the strips treated with the swollenin broth have asignificantly lower maximal load (about 15%) than the strips treatedwith control broth. The only difference between these two broths is thatone is from the fermentation of the yeast strain containing the T.reesei swollenin gene, while the control strain does not contain thisgene. These results show that there is a component in the swolleninbroth which is weakening filter paper.

EXAMPLE 9 Treatment of Cotton Fibers with Swollenin

[0121] The yeast clones described above in Example 4 were grown underthe conditions specified and the fermentation broth separated fromextraneous cell matter and debris. A control clone of yeast, whichcontained the expression plasmid but without the inserted swolleninencoding cDNA sequence, was also grown under the same conditions and thefermentation broth isolated by removing extraneous cell matter anddebris. The culture supernatants from two fermentations, one containingyeast transformed with the swollenin gene and one containing yeasttransformed without the swollenin gene as a control, were concentratedapproximately 50 fold and were used to determine the effects ofincubating T. reesei swollenin with cotton fibers. The effects of thetwo supernatants were further compared with the cellobiohydrolase I(CBHI) for T. reesei.

[0122] Mercerized cotton fibers were suspended in buffer (50 mM sodiumacetate, pH 5.0) containing supernatant from the yeast fermentations(dilution 1:4) and CBHI (dosage 5 μg/g). After incubation for 240minutes at 25° C., the suspended fibers were filtered off and the amountof reducing sugars released into the filtrates was determined by themethod of Sumner and Somers (1944). The fibers were rinsed once withbuffer and then suspended in distilled water with glass beads prior tosonication for one minute using a probe tip sonicator (Vibra Cell Sonicsand Materials Inc.) The fibers were then stained and visualized by lightmicroscopy to determine gross affects on their structure. The filtratefrom the control treatment and the filtrate originating from the yeaststrain containing the swollenin gene did not exhibit hydrolyticactivity, that is, no reducing sugars were liberated from the cottonfibers. In contrast, CBHI alone liberated reducing sugars 0.08% (oforiginal dry weight). Prior to sonication no difference between fiberstreated with supernatant from the control yeast strain versus fiberstreated with supernatant from the yeast strain containing the swolleningene could be discerned. However, after sonication swollen anddisorganized regions were apparent in fibers treated with supernatantfrom the yeast containing the swollenin gene which were not present inthe fibers treated with supernatant obtained from the control yeaststrain (FIG. 5). CBHI alone caused light fibrillation on the fibers, butno opened and swollen regions, which were typical effects forsupernatant from yeast containing the swollenin gene, were detected.

We claim:
 1. A partially or wholly isolated swollenin protein derivedfrom a microbial source.
 2. The swollenin protein according to claim 1,wherein said swollenin is derived from a fungus or bacteria.
 3. Theswollenin protein according to claim 2, wherein said fungus is afilamentous fungus.
 4. The swollenin protein according to claim 3,wherein said filamentous fungus is Trichoderma spp.; Humicola spp.,Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., orGliocladium spp.
 5. The swollenin according to claim 1, wherein saidswollenin comprises a sequence having at least 70% sequence identitywith the sequence provided in SEQ. ID NO:2 or comprises a derivative ofthe sequence according to SEQ. ID NO:2, wherein said swollenin furtherhas the ability to weaken filter paper and/or swell cotton fibers. 6.The swollenin protein according to claim 5, wherein said swollenincomprises a sequence consisting essentially of the sequence provided inSEQ. ID:NO
 2. 7. A DNA encoding a swollenin protein according toclaim
 1. 8. The DNA according to claim 7, wherein said DNA is derivedfrom a fungus or bacteria.
 9. The DNA according to claim 8, wherein saidfungus is a filamentous fungus.
 10. The DNA according to claim 9,wherein said filamentous fungus is Trichoderma spp., Huricola spp.,Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., orGliocladium spp.
 11. The DNA according to claim 7, wherein said DNAcomprises a sequence having at least 70% sequence identity with thesequence provided in SEQ. ID NO:1 or comprises a derivative of thesequence according to SEQ. ID NO:1, wherein said DNA encodes a swolleninprotein which has the ability to weaken filter paper and/or swell cottonfibers.
 12. A DNA according to claim 7, wherein said DNA hybridizes witha DNA having all or part of the sequence provided in SEQ ID NO:1.
 13. ADNA according to claim 12, wherein said DNA hybridizes with a DNA probeencoding a peptide having an amino acid sequence comprising SEQ. IDNO:14, SEQ. ID NO:15, SEQ. ID NO:16, SEQ. ID NO:17 or SEQ. ID NO:18. 14.A DNA comprising the sequence according to SEQ. ID NO:1.
 15. A vectorcomprising the DNA of claim
 7. 16. A host cell transformed with thevector of claim
 15. 17. The host cell according to claim 16, whereinsaid host cell has been genetically altered to delete one or morecellulase genes.
 18. The host cell according to claim 16, wherein saidhost cell is a filamentous fungus.
 19. The host cell according to claim18, wherein said filamentous fungus is Trichoderma spp., Humicola spp.,Neurospora spp., Aspergillus spp., or Fusarium spp.
 20. A method ofproducing swollenin protein comprising the steps of: (a) obtaining ahost cell which has been transformed with a vector comprising DNAencoding an swollenin protein, said DNA being isolated from a fungus orbacteria; (b) culturing said host cell under conditions suitable for theexpression and, optionally, secretion, of said swollenin protein; (c)recovering said fermentation broth containing said swollenin protein.21. The method according to claim 20, wherein said DNA is derived from afilamentous fungus.
 22. The method according to claim 21, wherein saidfilamentous fungus is Trichoderma spp., Humicola spp., Neurospora spp.,Aspergillus spp., Fusarium spp., or Gliocladium spp.
 23. The methodaccording to claim 20, wherein said DNA comprises a sequence having atleast 70% sequence identity with the sequence provided in SEQ. ID NO:1or comprises a derivative of the sequence according to SEQ. ID NO:1,wherein said DNA encodes a swollenin protein which has the ability toweaken filter paper and/or swell cotton fibers.
 24. The method accordingto claim 20, wherein said DNA hybridizes with a DNA having all or partof the sequence provided in SEQ ID NO:1.
 25. The method according toclaim 20, wherein said DNA hybridizes with a DNA probe encoding apeptide having an amino acid sequence comprising SEQ. ID NO:14, SEQ. IDNO:15, SEQ. ID NO:16, SEQ. ID NO:17 or SEQ. ID NO:18.
 26. The methodaccording to claim 20, wherein said DNA comprises the sequence accordingto SEQ. ID NO:1.
 27. A method of altering the properties of a cellulosicsubstrate comprising contacting said cellulosic substrate with acomposition comprising swollenin protein produced according to themethod in claim
 20. 28. The method according to claim 27, wherein saidmethod comprises altering the nutritional properties of an animal feed.29. The method according to claim 27, wherein said method comprisesaltering the properties of a fabric or yarn comprising cellulosicfibers.
 30. The method according to claim 27, wherein said methodcomprises altering the properties of wood pulp or derivatives thereofduring the manufacture of paper.
 31. The method according to claim 27,wherein said method comprises altering the properties of cellulosicbiomass during its reduction to glucose.
 32. The method according toclaim 27, wherein said method comprises altering the properties ofcellulosic corn husk fiber during its reduction to glucose.
 33. A methodof preparing a cellulase composition which is free of a swollenincomprising: (a) obtaining a microorganism which produces cellulase andswollenin; (b) treating said microorganism in a manner so as to disrupt,delete and or interfere with the expression of said swollenin protein;(c) culturing said microorganism under suitable conditions to expresscellulase; (d) collecting said expressed cellulase which lacks aswollenin protein.
 34. An animal feed comprising the swollenin ofclaim
 1. 35. A laundry detergent or textile treatment compositioncomprising the swollenin of claim 1.